The present invention relates to a whole cell assay for cathepsin K. The present invention is useful for determining cathepsin K activity in mammalian cell systems and for identifying and evaluating inhibitors of cathepsin K.
A variety of disorders in humans and other mammals involve or are associated with abnormal bone resorption. Such disorders include, but are not limited to, osteoporosis, glucocorticoid induced osteoporosis, Paget""s disease, abnormally increased bone turnover, periodontal disease, tooth loss, bone fractures, rheumatoid arthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma. One of the most common of these disorders is osteoporosis, which in its most frequent manifestation occur in postmenopausal women. Osteoporosis is a systemic skeletal disease characterized by a low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Osteoporotic fractures are a major cause of morbidity and mortality in the elderly population. As many as 50% of women and a third of men will experience an osteoporotic fracture. A large segment of the older population already has low bone density and a high risk of fractures. There is a significant need to both prevent and treat osteoporosis and other conditions associated with bone resorption. Because osteoporosis, as well as other disorders associated with bone loss, are generally chronic conditions, it is believed that appropriate therapy will typically require chronic treatment.
Osteoporosis is characterized by progressive loss of bone architecture and mineralization leading to the loss in bone strength and an increased fracture rate. The skeleton is constantly being remodeled by a balance between osteoblasts that lay down new bone and osteoclasts that breakdown, or resorb, bone. In some disease conditions and advancing age the balance between bone formation and resorption is disrupted; bone is removed at a faster rate. Such a prolonged imbalance of resorption over formation leads to weaker bone structure and a higher risk of fractures.
Bone resorption, is primarily performed by multinuclear giant cells, the osteoclasts. The mechanism by which osteoclasts resorb bone is by an initial cellular attachment to bone tissue followed by the formation of an extracellular compartment or lacunae. The lacunae are maintained at a low pH by a proton-ATP pump. The acidified environment allows for initial demineralization of bone followed by the degradation of bone proteins or collagen by proteases such as cysteine proteases (Delaisse, J. M. et al., 1980, Biochem J 192:365-368; Delaisse, J. et al., 1984, Biochem Biophys Res Commun:441-447; Delaisse, J. M. et al.,1987, Bone 8:305-313). Collagen constitutes 95% of the organic matrix of bone. Therefore, proteases involved in collagen degradation are an essential component of bone turnover, and the development and progression of osteoporosis.
Cathepsins belong to the papain superfamily of cysteine proteases. These proteases function in the normal physiological as well as pathological degradation of connective tissue. Cathepsins play a major role in intracellular protein degradation and turnover and remodeling. To date, a number of cathepsin have been identified and sequenced from a number of sources. These cathepsins are naturally found in a wide variety of tissues. For example, cathepsin B, F, H, L, K, S, W, and Z have been cloned. Cathepsin K (which is also known by the abbreviation cat K) is also known as cathepsin O and cathepsin O2. See, PCT Application WO 96/13523, Khepri Pharmaceuticals, Inc., published May 9, 1996.
Cysteine protease inhibitors such as E-64 (trans-epoxysuccinyl-L-leucylamide-(4-guanidino) butane) are known to be effective in inhibiting bone resorption (Delaisse, J. M. et al., 1987, Bone 8:305-313). Recently, cathepsin K was cloned and found specifically expressed in osteoclasts (Tezuka, K. et al., 1994, J Biol Chem 269:1106-1109; Shi, G. P. et al.,1995, FEBS Lett 357:129-134; Bromme, D. and Okamoto, K., 1995, Biol Chem Hoppe Seyler 376:379-384; Bromme, D. et al., 1996, J Biol Chem 271:2126-2132; Drake, F. H. et al., 1996, J Biol Chem 271:12511-12516). Concurrent to the cloning, the autosomal recessive disorder, pycnodysostosis, characterized by an osteopetrotic phenotype with a decrease in bone resorption, was mapped to mutations present in the cathepsin K gene. To date, all mutations identified in the cathepsin K gene are known to result in inactive protein (Gelb, B. D. et al., 1996, Science 273:1236-1238; Johnson, M. R. et al., 1996, Genome Res 6:1050-1055. Therefore, it appears that cathepsin K is probably involved in osteoclast mediated bone resorption.
Cathepsin K is synthesized as a 37 kDa pre-pro enzyme, which is localized to the lysosomal compartment and where it is presumably autoactivated to the mature 27 kDa enzyme at low pH (McQueney, M. S. et al., 1997, J Biol Chem 272:13955-13960; Littlewood-Evans, A. et al., 1997, Bone 20:81-86). Cathepsin K is most closely related to cathepsin S having 56% sequence identity at the amino acid level. The S2P2 substrate specificity of cathepsin K is similar to that of cathepsin S with a preference in the P1 and P2 positions for a positively charged residue such as arginine, and a hydrophobic residue such as phenylalanine or leucine, respectively (Bromme, D. et al, 1996, J Biol Chem 271: 2126-2132; Bossard, M. J. et al.,1996,) J Biol Chem 271:12517-12524). Cathepsin K is active at a broad pH range with significant activity between pH 4-8, thus allowing for good catalytic activity in the resorption lacunae of osteoclasts where the pH is about 4-5.
Human type I collagen, the major collagen in bone is a good substrate for cathepsin K (Kafienah, W., et al, 1998, Biochem J 331:727-732). In vitro experiments using antisense oligonucleotides to cathepsin K, have shown diminished bone resorption in vitro probably due to a reduction in translation of cathepsin K mRNA (Inui, T., et al., 1997, J Biol Chem 272:8109-8112. The crystal structure of cathepsin K has been resolved (McGrath, M. E., et al., 1997, Nat Struct Biol 4:105-109; Zhao, B., et al., 1997, Nat Struct Biol 4: 109-11) and selective peptide based inhibitors of cathepsin K have been developed (Bromme, D., et al., 1996, Biochem J 315:85-89; Thompson, S. K., et al., 1997, Proc Natl Acad Sci U S A 94:14249-14254). Analyses of these inhibitors should provide further evidence for the role of cathepsin K in bone resorption and in pathological disorders such as osteoporosis.
An in vitro model for bone resorption, is a pit formation assay in which purified osteoclasts are cultured on bone slices and resorbing activity is determined by measuring collagen degradation products using ELISA or by counting the number of pits formed (Nesbitt, S. A., and Horton, M. A., 1997, Science 276:266-269; Salo, J., et al., 1997, Science 276:270-273). It is thought that the cultured osteoclasts create tight junctions with bone matrix forming lacunae in which collagen is degraded, the degraded collagen is transcytosed through the osteoclasts. Drawbacks of this assay are; does not specifically measure cathepsin K activity, isolation of osteoclasts is long and difficult, quantitation of pits is variable and changes in pit area is not assessed.
In U.S. Pat. No. 5,871,946, there is described a method for determining the activity of enzymes in metabolically active whole cells in the absence of any genetic manipulations for the purpose of identifying abnormal cells. The enzymes described include different cathepsins. The patent teaches different reagents to be used for enzymatic substrates and the methods for making these reagents. However, this patent does not teach a specific genetically engineered cell-based assay for cathepsin K as a tool for identifying cell-permeable inhibitors of cathepsin K.
To date there are no known cell-based assays that specifically determine cathepsin K activity that can be used to identify cell-permeable inhibitors of cathepsin K and are amenable to high throughput screening. Therefore, one object of the present invention is to develop a cell-based assay specific to cathepsin K and devoid of the activity of other proteases. An additional object is to provide a cell-based assay specific to cathepsin K that can be easily scaled up for high throughput screening for screening and identifying inhibitors to cathepsin K. To that end, a stable cell line expressing cathepsin K and in which the pre-pro- enzyme is intracellularly processed to its mature and active form is provided.
The invention further provides cell permeable cathepsin K substrates, useful in measuring intracellular cathepsin K activity.
Another object is to provide a reliable and reproducible assay for cathepsin K using conditions in which extracellular protease activity is inhibited.
A further object of the present invention is to provide an assay for cathepsin K under reaction conditions favoring the measurement of intracellular cathepsin K activity. Such an assay can be scaled up for high throughput screening to identify inhibitors to cathepsin K.
These and other objects will become apparent to those of ordinary skill from the teachings provided herein.
The application refers to a number of publications, patents and patent applications the contents of which are hereby incorporated by reference in their entirety.
In one embodiment, the present invention relates to a method for assaying cathepsin K activity in a mammalian host cell, comprising the steps of:
a) transforming a host cell with a vector containing a nucleotide sequence encoding pre-pro-cathepsin K to provide a transformed host cell;
b) incubating said transformed host cell with a substrate to said cathepsin K; and
c) determining the cathepsin K activity in the transformed host cell.
Another embodiment of the present invention relates to a screening method for identifying a compound useful as an inhibitor of cathepsin K comprising the steps of:
a) incubating an eukaryotic host cell transformed with a vector containing a nucleotide sequence encoding pre-pro-cathepsin K with a substrate to the cathepsin K in the presence of said compound;
b) incubating an eukaryotic host cell transformed with a vector containing a nucleotide sequence encoding pre-pro-cathepsin K with a substrate to said cathepsin K in the absence of said compound;
c) comparing the cathepsin K activity of (a) to (b).
The present invention is further directed to expression vectors comprising a nucleotide sequence encoding cathepsin K and to recombinant host cells and cell lines comprising the said vector. The present invention further includes, assay kits for determining cathepsin K activity and high throughput screening systems for identifying inhibitors of cathepsin K.
All percentages and ratios used herein, unless otherwise indicated, are by weight. The invention hereof can comprise, consist of, or consist essentially of the essential as well as optional ingredients, components, and methods described herein.